Addendum - ISSC proposal...Proposal No. 13-118-S 2013 Task Force I – Proposal No. 13-118-S - Page 2 of 13 (3) Bacteriological examination does not ordinarily show the presence of

Interstate Shellfish

Sanitation Conference

Addendum to

Proposals for Consideration

at the

2013 Biennial Meeting

January 25 – January 31, 2014

The St. Anthony Riverwalk Hotel

“a national historic landmark”

2013 Proposal Inventory

Substitute Proposals, Additional Information and Late Proposals

Proposal

Number

Submitter / Proposal Subject

Page

Number

13-101-S ISSC Executive Office

Outbreaks of Shellfish Related Illnesses

Substitute Proposal

1

13-118-S US Food & Drug Administration (Greg Goblick)

Dilution Guidance for Prohibited Zones Associated with Wastewater Discharges

4

13-119-L

Laboratory Methods Review & Quality Assurance Committee (Patti Fowler)

Revisions to Model Ordinance Chapter III. Laboratory

Requirements for the Authority

17

13-120-L Spinney Creek Shellfish, Inc. (Tom Howell)

Male-Specific Coliphage Method for Quahogs (M. mercenaria)

20

13-202-S Ken Moore (ISSC)

Requirements for Outbreaks of Shellfish Related Illnesses

Substitute Proposal

49

13-204-I US Food & Drug Administration (Paul DiStefano)

Vibrio Control Plans

Additional Public Health Rationale

52

13-216-S Pacific-Rim Shellfish Sanitation Conference (Kenichi Wiegardt)

Shellstock Storage Critical Control Point Exemption for Panopea generosa as

Species

60

13-218-S Borough of Highlands Depuration Commission (Larry Colby)

Accounting of Shellfish Quantities in Depuration Facilities

61

13-221-L US Food & Drug Administration (Paul DiStefano)

Vibrio parahaemolyticus Control Plan for Hard Clams

64


Vibrio parahaemolyticus Control Plan Water Temperatures

67


Vibrio parahaemolyticus Control Plan Risk per Serving

70

13-224-L ISSC Executive Board

Implementation Date for Harvester and Dealer Training Requirements

73

13-225-L US Food & Drug Administration (Melissa Evans)

Guidance for Submission of Post-Harvest Processing Validation Studies

74

13-226-L US Food & Drug Administration (Melissa Evans)

Guidelines for Primary Certified Shellfish Processors on Using Controls for

Irradiation of Containers of Molluscan Shellfish Pre-Labeled with Vibrio

Reduction Language

77

13-227-L Spinney Creek Shellfish, Inc. (Lori Howell)

Eliminate Requirements for the Authority to Retain Records of a Trade Secret

or Proprietary Nature

80

Key:

I = Additional Information

L = Late Proposal

S = Substitute Proposal

Substitute Language

Proposal No. 13-101

Proposal No. 13-202

_____________________________________________________________________________________

2013 Task Force I & II – Substitute Language Proposals 13-101 and 13-202 - Page 1 of 3

NSSP Guide for the Control of Molluscan Shellfish

Section I. Model Ordinance

Chapter II. Risk Assessment and Risk Management

@.01 Outbreaks of Shellfish Related Illness

Insert New Section:

F. When the investigation outlined in Section @.01 A. indicates the illness(es) are

associated with the naturally occurring pathogen Vibrio parahaemolyticus (V.p.) , the

Authority shall determine the number of cases epidemiologically associated with

implicated area and actions taken by the Authority will be based on the number of cases

and the span of time as follows.

(1) When sporadic cases do not exceed a risk of one (1) illness per 100,000 servings or involves at least two (2) but not more than four (4) cases occurring within a

thirty (30) day period from a hydrologically connected water body in which no

two (2) cases occurred from a single harvest day, the Authority shall:

(a) Determine the extent of the hydrologically connected water body, and

(b) Issue a consumer advisory for all shellfish (or species implicated in the

illness) from the implicated area; and

(c) Notify receiving States, the ISSC and the FDA Regional Shellfish

Specialist that a potential health risk is associated with shellfish harvested

from the implicated growing area, and

(2) When the risk exceeds one (1) illness per 100,000 servings within a thirty (30)

day period or when cases exceed four (4) but not more than ten (10) over a thirty

(30) day period from a hydrologically connected water body and when two (2) or

more cases but less than four (4) cases occur from a single harvest day, the

Authority shall:

(a) Determine the extent of the hydrologically connected water body; and

(b) Issue a consumer advisory for all shellfish (or species implicated in the

illness) from the implicated growing area; and

(c) Immediately place the implicated portion(s) of the harvest area(s) in the

closed status; and

(d) Notify receiving States, the ISSC, and the FDA Regional Shellfish

Specialist that a potential health risk is associated with shellfish harvested

from the implicated growing area; and

(e) As soon as determined by the Authority, transmit to the FDA and

receiving States information identifying the dealers shipping the

implicated shellfish.

[Proposal Addendum Page 1 of 80]

Substitute Language

Proposal No. 13-101

Proposal No. 13-202

_____________________________________________________________________________________


(3) When the number of cases exceeds ten (10) illnesses within a thirty (30) day

period from a hydrologically connected growing area or four (4) cases occurred

from a single harvest date, The Authority shall:

(a) Determine the extent of the hydrologically connected water body; and (b) Immediately place the implicated portion(s) of the harvest area(s) in the

closed status; and

(c) Promptly initiate a voluntary industry recall consistent with the Recall

Enforcement Policy, Title 21 CFR Part 7. The recall shall include all

implicated products.

(4) When a growing area has been closed as a result of V.p. cases, the Authority shall

keep the area closed for the following periods of time to determine if additional

illnesses have occurred:

(a) The area will remain closed for a minimum of seven (7) days when sporadic cases do not exceed a risk of one (1) illness per 100,000 servings

or involves four (4) or less cases occurring within a thirty (30) day period

from a hydrologically connected water body in which no two (2) cases

occurred from a single harvest date.

(b) The area will remain closed for a minimum of fourteen (14) days when the risk exceeds one (1) illness per 100,000 servings within a thirty (30) day

period or cases exceed four (4) but not more than ten (10) cases over a

thirty (30) day period from a hydrologically connected water body with

two (2) or more cases but less than four (4) cases occurring from a single

harvest date.

(c) The area will remain closed for a minimum of twenty-one (21) days when the number of cases exceeds ten (10) illnesses within thirty (30) days or

four (4) cases occur from a single harvest date from a hydrologically

connected growing area,

(5) Prior to reopening an area closed as a result of V.p. cases, the Authority shall:

(a) Collect and analyze samples to ensure that tdh does not exceed 10/g and

trh does not exceed 10/g; or

(b) Ensure that environmental conditions have returned to levels not

associated with V.p. cases.

(6) Shellfish harvesting may occur in an area closed as a result of V.p. illnesses when

the Authority implements one or more of the following controls:

(a) Post harvest processing using a process that has been validated to achieve

a two (2) log reduction in the levels of total Vibrio parahaemolyticus for


Substitute Language

Proposal No. 13-101

Proposal No. 13-202

_____________________________________________________________________________________


Gulf and Atlantic Coast oysters and a three (3) log reduction for Pacific

Coast oysters;

(b) Restricting oyster harvest to product that is labeled for shucking by a

certified dealer, or other means to allow the hazard to be addressed by

further processing;

(c) Limiting the time to one (1) hour from harvest to an internal temperature

of 50°.

(d) Other control measures that based on appropriate scientific studies are

designed to ensure that the risk of V.p. illness is no longer reasonably

likely to occur, as approved by the Authority.


Proposal No. 13-118-S

2013 Task Force I – Proposal No. 13-118-S - Page 1 of 13

Proposal for Task Force Consideration at the

Interstate Shellfish Sanitation Conference


Growing Area

Harvesting/Handling/Distribution

Administrative

Submitter: Food and Drug Administration

Affiliation: Food and Drug Administration

Address:

Center for Food Safety and Applied Nutrition

5100 Paint Branch Parkway

College Park, MD 20740

Phone: 240-402-2768

Fax: 240-402-2601

Email:

[email protected]

Proposal Subject: Dilution Guidance for Prohibited Zones Associated with Wastewater Discharges

Specific NSSP

Guide Reference:

NSSP Guide Section IV. Guidance Documents

Chapter II. Growing Areas

Text of Proposal/

Requested Action

.16 Determining Appropriately Sized Prohibited Areas Associated with

Wastewater Treatment Plants

Introduction

Molluscan shellfish are filter feeders and therefore have the ability to concentrate

microorganisms from the water column, including human pathogens and toxigenic

micro-algae if these organisms are present. Concentrations of microorganisms in the

shellfish may be as much as 100 times greater than those found in the water, and if the

microorganisms are harmful to humans, illness can result. The correlation between

sewage pollution of shellfish waters and illness has been demonstrated many times.

Certain shellfish-borne infectious diseases are transmitted via the fecal-oral route,

with the cycle beginning with the fecal contamination of the shellfish growing waters.

In the winter of 1924-25, an oyster-borne typhoid outbreak occurred in the United

States which caused a large number of illnesses and deaths (Lumsden, et al 1925). In

response to this outbreak the National Shellfish Sanitation Program (NSSP) was

initiated by the States, the U.S. Public Health Service, and the shellfish industry.

Research at the time indicated that typhoid fever would not ordinarily be attributed to

shellfish harvested from water in which not more than 50% percent of the one cc (ml)

portions of water examined were positive for fecal coliform bacteria (an MPN of

approximately 70 per 100 ml), provided that the areas were not subject to direct

contamination with small amounts of fresh sewage which would not likely be

revealed by routine bacteriological examination. As a result water quality criteria

were established, namely;

(1) The area be sufficiently removed from major sources of pollution so that the shellfish are not subjected to fecal contamination in quantities which might be

dangerous to public health;

(2) The area be free from pollution by even small quantities of fresh sewage;




(3) Bacteriological examination does not ordinarily show the presence of the coli-aerogenes group of bacteria in one cc dilution of the growing area water.

Once these standards were adopted in the United States in 1925, reliance on these

criteria for evaluating the safety of shellfish harvesting areas has generally proven

effective in preventing major outbreaks of disease transmitted by the fecal-oral route.

Today, fecal and total coliforms are used as an index of the sanitary quality of a

growing area and to foretell the possible presence of fecal transmitted bacterial

pathogens. The goal of the NSSP remains the same – to ensure the safety of shellfish

for human consumption by preventing harvest from contaminated growing areas.

However, there is now ample scientific evidence to show that the current bacterial

indicators are inadequate to predict the risk of viral illness for the following reasons:

(1) Enteric viruses are resistant to treatment and disinfection processes in a wastewater treatment plant (WWTP) and are frequently detected in the WWTP’s

final effluent under normal operating conditions (Baggi et al. 2001; Burkhardt et

al. 2005).

(2) Shellfish can bioaccumulate enteric viruses up to 100-fold from surrounding water (Seraichekas et al. 1968; Maalouf et al. 2011).

(3) Certain enteric viruses are retained by molluscan shellfish to a greater extent and for longer than the indicator bacteria currently used to classify shellfish growing

areas (Sobsey et al. 1987; Dore & Lees 1995; Love et al. 2010). It has been well

documented that enteric virus detection is not indexed by levels of conventional

indicator bacteria.

For several decades now viral illnesses (in particular norovirus (NoV) and Hepatitis A

(HAV)) have been the most common food safety problem associated with bivalve

molluscan shellfish (Woods & Burkhardt. 2010; Iwamoto et al 2010; Scallan et al.

2011; Batz et al. 2012). NoV genogroups I, II and IV and HAV are human specific

and transferred by the fecal-oral route. Because WWTPs do not completely remove

infectious enteric viruses emphasis should be placed on the importance of ensuring

there is adequate dilution between a sewage source and a shellfish growing area.

The purpose of this guidance is to provide the scientific basis and recommendations

for determining appropriately sized Prohibited Areas (closure zones) based on the

minimum criteria established under Section II, Chapter IV. @.03 E(5) of the Model

Ordinance (Section E Prohibited Classification).

Classification Requirements for Growing Areas Associated with Waste Water

Treatment Plants

The NSSP Model Ordinance (MO) requires that a comprehensive sanitary survey be

undertaken prior to the classification of the growing area as Approved, Conditionally

Approved, Restricted, or Conditionally Restricted.

The sanitary survey must take careful recognition of any WWTPs as they represent

one of the major sources of human sewage pollution. It is preferable that the shellfish

growing areas be sited so far away from sewage discharges that the WWTP effluent




has no hazardous effect, because there is a direct relationship between the level of

WWTP effluent dilution and the level of enteric viruses detected in the shellfish

(Goblick et al. 2011).

Delineation of the Prohibited Zone around a Wastewater Treatment Plant

The NSSP MO Section II, Chapter IV. @.03 (2) (b) states that all growing areas

which have a sewage treatment plant outfall or other point source outfall of public

health significance within or adjacent to the shellfish growing area shall have a

prohibited classification established adjacent to the outfall taking account of the

following factors:

(1) The volume flow rate, location of discharge, performance of the wastewater treatment plant and the bacteriological or viral quality of the effluent;

(2) The decay rate of the contaminants of public health significance in the wastewater discharged;

(3) The wastewater's dispersion and dilution and the time of waste transport to the area where shellstock may be harvested; and

(4) The location of the shellfish resources, classification of adjacent waters and identifiable landmarks or boundaries.

There are several important considerations for the shellfish authority to consider when

establishing the size of the prohibited zone:

(1) The distance to ensure that there is adequate dilution when the WWTP is operating as normal. “Normal” means that the WWTP is operating fully within

the plant’s design specifications, including design flows, treatment stages,

disinfection, as well as compliance with all permit conditions.

If the plant is operating outside of the normal parameters it shall be considered to

be malfunctioning.

(2) That the collection system has no malfunctions, bypasses or other factors that would lead to significant sewage leakages to the marine environment.

(3) That there is adequate time when any malfunction occurs to ensure that all harvesting ceases and closures are enforced, so that contaminated product does

not reach the market.

The following guidelines shall be used when assessing these factors in the dilution

analysis for the closure zone:

1) Volume flow rate: For a minimally sized prohibited zone for Conditionally Approved areas managed in part based on the performance of the WWTP, the

maximum monthly average flow at the WWTP recorded in the Monthly

Operating Reports (MORs) maintained by the WWTP permitting authority should

be used considering at a minimum the most recent two years of flow records. If

the maximum monthly average flow at the WWTP from two consecutive years of

flow records is within 85 – 100% of the design flow, then the design flow should




be used. Thus, these flow values are appropriate when establishing a minimally

sized prohibited zone when the WWTP is considered to be operating under

normal operating conditions.

Additional information and historical data may be accessed on the U.S.

Environmental Protection Agency (EPA) website at:

http://cfpub.epa.gov/dmr/index.cfm. Consistent with the EPA regulations in 40

CFR 122.2, the maximum monthly average flow, which is typically reported in

the MOR, is defined as the average ‘‘daily discharges’’ over a calendar month,

calculated as the sum of all ‘‘daily discharges’’ measured during a calendar

month divided by the number of ‘‘daily discharges’’ measured during that month

typically expressed in units of million gallons per day (MGD). Thus, the

maximum monthly average flow is defined as the highest average monthly flow

(MGD) within at a minimum the most recent consecutive two years of flow

records. The design flow is defined as the flow (MGD) that the WWTP is

designed to discharge and can be expressed as a daily, monthly, or annual

discharge. In the design of WWTPs, various flow regimes are considered such as

the average flow, maximum flow and peak (instantaneous) flow. However, it is

important to note that certain tolerances are allowed under EPA NPDES program

and WWTPs are not necessarily expected to meet permit conditions over all flow

regimes. Thus, if permit limits are expressed as a monthly average it is

considered acceptable for the permitted pollutants to exceed the permit on a short

term basis as long as the permit condition (monthly average) is met. It is also

important to note that EPA does not have any permit limitations established for

the discharge of viruses.

In the context of public health, some of these flow regimes such as when

average hourly flows exceed the design flow can be associated with

periods of effluent degradation leading to an increase in the viral load in

the effluent. Utilizing average hourly flows and comparing against the

design flow ensures that the periods when effluent degradation are most

likely to occur are adequately identified and assessed. Average hourly flow

rates within the most recent two years of records should be evaluated to

assess the likelihood that the average hourly flows can exceed the design

flow. In the absence of supporting data, the conditional area should be

closed when the average hourly flow rates exceed the WWTP design flow

due to the potential degradation of the virological quality of treatment.

FDA studies have determined that when WWTP average hourly flow rates

exceed design flow the virological quality of effluent typically degrades

beyond what is considered as normal treatment. Moreover, FDA

bioaccumulation studies indicate that shellfish can accumulate significant

levels of viral pathogens when exposed in durations of less than one hour.

However, a flow level threshold above the design flow could be

determined on a case by case basis provided the virological quality of the

effluent is assessed. The average hourly flow is defined as the average flow

measured over an hour. More detailed flow records are typically

maintained and can be accessed through the permitted WWTP.


http://cfpub.epa.gov/dmr/index.cfm



When conditional management based on WWTP performance is not employed

the prohibited zone shall be sufficient in size to dilute the microbial loadings

resulting from a WWTP malfunction (such as a sewage bypass or a loss of

disinfection) to ensure the Approved area adjacent to the prohibited zone will

meet the bacteriological standards for Approved area classification under all

conditions including a WWTP malfunction. If the WWTP has no prior history of

sewage bypasses then at a minimum a loss of disinfection malfunction shall be

considered when sizing the prohibited zone. As many WWTP malfunctions

occur from hydraulic overloading as a result of rainfall, snowmelt, storm events

or periods of high flow, a maximum average hourly rate shall be considered when

determining the size of the prohibited zone. The maximum average hourly flow

is defined as the highest average hourly flow recorded within at a minimum) the

most recent two consecutive years of flow records.

Location of discharge: The location of the discharge must be determined in

order to define the distance from the point of effluent discharge to shellfish

growing areas that could be impacted. The distance from shore and the depth of

the WWTP outfall also can be used in the dilution analysis of the discharge. The

location of discharge includes the location, number, size and orientation of the

discharge port(s) on the outfall or its diffuser.

When determining if a WWTP within the watershed or catchment area draining to

a shellfish estuary potentially impacts a shellfish growing area, in the absence of a

database collected, the NSSP recommends that a worst case raw sewage

discharge be assumed. In this circumstance a level of 1.4 x 106 FC/100ml

assumed for a raw sewage release-requires a 100,000:1 dilution to dilute the

sewage sufficient to meet the approved area standard of 14 FC/100ml. If dilution

analysis determines that the location of the discharge is such that the dilution of

effluent would be greater than 100,000:1 then the WWTP could be considered

located outside the zone of influence to the shellfish growing area. A lower

dilution level could be justified provided that specific data to that particular

WWTP demonstrates that a lower bacteriological level associated with a potential

raw sewage discharge is supported. Additional or other site specific information

also can be used to justify alternative approaches that may take into account other

factors (such as no prior history of raw sewage discharges or containment

structures sufficiently sized to accommodate a raw sewage event preventing a

discharge).

It should also be noted that if shellfish harvesting occurs within the zone of

influence from a WWTP then these areas are subject to a WWTP Management

Plan as defined in Section II Chapter IV @. 03 C.(2)(a) of the MO. Additionally,

if a departure of the normal WWTP function could potentially impact a shellfish

growing area then the areas affected should be managed under a conditional

management plan as defined in Section II Chapter IV @. 03 C.(2)(a) of the MO.

The minimum size of a prohibited zone for a conditional area under a WWTP

management plan should be determined considering both the minimum dilution




(1000:1) needed to mitigate the presence of viruses in treated effluent (or a

scientifically based alternative approach) as well as the prerequisite notification

time to close the conditional area during a WWTP malfunction or period of

degraded effluent quality, prior to the conditional area receiving the impact from

the WWTP effluent.

Performance of the WWTP: When considering the present and past performance

of the WWTP, this review should include information regarding the wastewater

collection system, inspection of essential plant components (including any

monitoring and alarm systems), events whereby the plant exceeds its design

capacity and an evaluation of the disinfection system. The plants past

performance should also include a file review of the plant’s Discharge Monitoring

Reports, considering at a minimum, the most recent two years of permit records.

When there is evidence that the WWTP exceeds design capacity, consideration

should then be given to the frequency of such events and the effect this will have

on the plant’s ability to reduce the viral load of the effluent.

Consideration should also be given to the frequency of which the WWTP

bypasses any stage of treatment or any condition that may degrade the quality of

the effluent to determine the potential frequency a conditional growing area may

need to close over the course of a year. This assessment will determine the

feasibility of operating a conditionally managed area based on WWTP

performance.

Bacteriological or viral quality of the effluent: Discharge Monitoring Reports for

WWTPs should be examined and periodically monitored to assess the reliability

of the disinfection systems. Any samples collected to assess the reliability of the

disinfection system should be collected during the period(s) of the year that the

State Shellfish Control Authority (SSCA) deems most likely to experience

adverse conditions in the treatment or disinfection processes that could affect

effluent quality impacting receiving waters.

Results from any bacteriological or viral sampling and analyses must be

correlated with WWTP operation and evaluated in terms of the minimum

treatment expected when there is a malfunction, overloading or other poor

operational condition. However, it is essential to recognize that water samples

collected near discharge outfalls are not useful for determining the size of

prohibited zones because normal operating conditions in WWTPs can effectively

reduce or even eliminate the fecal and total coliforms - the current indicator

microorganisms used to assess treatment efficiency. In contrast, many human

enteric viruses are not inactivated by functional WWTP systems, hence the need

for an adequate dilution zone between the outfall and the shellfish resource.

Decay rate of contaminants: It should be assumed that there is no fecal coliform

or viral inactivation in the effluent during possible upset conditions in the

WWTP. There are a number of conditions that affect bacterial and viral

inactivation, including temperature, exposure to sunlight and sedimentation levels

in the water (Burkhardt et al, 2000; Lees, 2002; LaBelle, 1980; Griffen, 2003).

Scientists are unsure how long viruses remain viable in the marine environment,

but it is likely to be weeks or months (Younger, 2002), and enteroviruses have




been found in marine sediments suggesting that these sediments can be a source

upon resuspension (Lewis, 1986). Moreover, molluscan shellfish have been

found to retain viruses to a greater extent and for much longer periods than they

do bacteria (Sobsey et al, 1987; Richards, 1988; Dore and Lees, 1995; Dore et al,

2000; Shieh et al, 2000).

Waste water dispersion and dilution: Dispersion of the effluent refers to the

spread, location, and shape of the discharge plume with time as it leaves the

WWTP outfall. Dilution of the effluent refers to the amount of receiving water

that is entrained within a particular time or distance from the outfall, e.g. the

dilution of the effluent within the time or distance it takes to reach the border of

the prohibited zone. A dye study can be used to measure the dilution and

dispersion of the effluent during specific discharge conditions. Computer

modeling programs can also be used to estimate the dispersion and dilution of the

effluent plume from WWTPs.

In poorly flushed estuaries and coastal embayments there is the potential for

WWTP effluent build-up that further reduces the availability of “clean” waters to

both dilute contaminant loadings and purge shellfish of contaminants (Goblick et

al., 2011).

Time of waste transport to the shellfish harvest site: When there is a WWTP

malfunction it is important that adequate systems are in place to officially close

the harvest area before the effluent impacts the shellfish. This is a mandatory

requirement for conditional management of shellfish harvest areas and all parties

must agree in writing on the process steps necessary to close the harvest area after

such events. Both time of travel and dilution should be considered when sizing a

prohibited zone around a WWTP outfall adjacent to a conditional growing area.

The overall sizing of the prohibitive zone should satisfy both a minimum dilution of 1000:1 and also factor in adequate time to respond to a malfunction event.

When establishing the time of travel between the WWTP and the classified area,

consideration should be given to the worst scenarios which would cause the

fastest travel. For example, the peak current flows at or near the outfall during

ebb tide and flood tide to determine effluent transport speeds. Current velocity

information may need to be generated if such information is not available or

adequate for the area of the outfall. Current velocity information can be obtained

from hydrographic dye studies, drogue studies, or current meter data conducted in

the vicinity of the outfall.

Location of shellfish resources: The best information that is available should be

used for locating shellfish resources near the outfall. Subtidal shellfish resources

may also be identified in sanitary surveys near WWTP outfalls. Therefore the

SSCA must establish closure zones at WWTP outfalls in accordance with the

classification requirements of the Model Ordinance..

Classification of Adjacent Waters: If the SSCA’s dilution analysis determines

that the shellfish water quality standards for approved waters are met at the

boundary of the prohibited area during potential upset conditions, the shellfish

area adjacent to the prohibited area need not be classified as Conditionally

Approved and may be classified as Approved.




Scientific Rationale for 1000:1 Dilution Guidance

Since 1987 FDA has recommended at training courses and other venues the use of a

1000:1 dilution as the minimum level of dilution needed around a WWTP outfall to

mitigate the impact of viruses for shellfish harvest areas managed conditionally based

on the performance of the WWTP. It has been advised that conditional management

based on WWTP performance may not be appropriate for all WWTP’s that are

located within proximity to shellfish harvest areas and recommended only for large,

highly efficient WWTPs that are well monitored.. In 1995 this estimated level of

necessary dilution was further calculated and explained by FDA using assumptions

based on the most relevant scientific literature available at that time (Kohn, et al.

1995; Havelaar et al. 1993; Kapikian et al. 1990; Liu et al. 1966). Since then major

advances in the detection and enumeration of NoV in wastewater and shellfish have

been made, and advances in fluorometer technologies have enabled more

sophisticated hydrographic dye study methods. Using these advances, FDA has

conducted dye studies supplemented with the testing of shellfish sentinels for enteric

viruses and their surrogates. This has afforded FDA for the first time with a means to

directly determine the viral risk posed by WWTP effluent on shellfish resources.

During recent years FDA has presented the findings from these studies at regional

shellfish meetings, at the biennial ISSC meeting, at international scientific

conferences and to international partners engaged in collaborative projects. Results

from these studies are referred to herein as part of the scientific basis for the current

recommended guidance.

In 2008 FDA performed an investigation in the upper portion of Mobile Bay,

Alabama, the results of which were published in the Journal of Shellfish Research

(Goblick, et al., 2011). The article describes how FDA used the aforementioned

technical advances to prospectively assess the 1995 1000:1 dilution estimate

recommendation and determine if this level of dilution is appropriate to mitigate the

risk of viruses discharged in treated wastewater effluent. From 2008 through 2012

FDA conducted four additional studies (Hampton Roads, Virginia; Yarmouth, Maine;

Coos Bay, Oregon; Blaine, Washington). In each of these studies, FDA evaluated

male-specific coliphage (MSC) and NoV levels in shellfish together with the dilutions

of WWTP effluent. The studies were designed to build a more comprehensive and in-

depth understanding of viral impacts posed by WWTPs on shellfish resources.

To date, findings from these studies demonstrate that achieving a steady-state 1000:1

dilution level in the requisite Prohibited area appears to be adequate for mitigating the

impacts of viruses on shellfish when WWTPs have typical treatment and disinfection

practices, such as secondary treatment and the use of chlorine, and when they are

operating under normal conditions. Results further indicate that in certain instances,

such as when WWTPs begin to exceed their design capacity, bypass treatment, or

otherwise malfunction, the 1000:1 dilution level may be inadequate and emergency

closure procedures should be considered within the conditional area management

plan. Under such circumstances, conditional area management plans should ensure

there is sufficient time for notification to the State Shellfish Control Authority

(SSCA) and for subsequent notifications closing the conditional area to harvesting.

MSC results in shellfish from the 2008-2012 studies were evaluated using 50

PFU/100 g as the threshold level of concern for MSC, since this is the level under the

Model Ordinance (Section II, Chapter IV, @.03 A(5)(c)(ii)) used for re-opening




harvest areas after an emergency closure due to raw untreated sewage discharged

from a large community sewage collection system or a WWTP. For conventional

WWTPs operating under normal conditions, there were at least four occasions when

dilution levels were between 700:1 and 1000:1 and MSC levels in shellfish exceeded

50 PFU/100g, but there were no occasions in which MSC levels exceeded 50

PFU/100g and dilution was greater than 1000:1. For conventional WWTPs operating

under malfunction conditions, such as when flow rates exceeded the design capacity

or during a treatment stage bypass, MSC levels in shellfish exceeded 50 PFU/100g in

at least 13 instances in which dilution was greater than 1000:1.

When evaluating the NoV results of the 2008 – 2012 studies FDA used a value of 300

RT-PCR units of NoV/100 gram of digestive gland (digestive diverticula) as the

threshold. This value was considered significant since at this level shellfish related

illnesses have been reported and demonstrated by the analysis of meal remnants.

In examining the results from all the studies, there were no cases in which

conventional WWTPs operating under normal conditions produced results greater

than 300 NoV particles/100 g of DD in oyster sentinels when dilution levels at the

associated sentinel stations were greater than 1000:1. When dilution levels were less

than 1000:1, levels of NoV GII greater than 300 NoV particles/100 g of DD were

detected, and on one occasion around 8000 NoV particles/100g DD were found.

On three occasions during which WWTPs were operating under malfunction

conditions (as previously described), thirteen (13) oyster samples were found with

NoV GII levels greater than 300 NoV particles/100 g DD when dilution was close to

or greater than 1000:1. These results emphasize the critical need for sufficient

notification time, meaning travel time from the WWTP discharge in Prohibited Area

is long enough to close the shellfish growing area in the event of a malfunction. This

preventative measure may necessitate the Prohibited Area be larger than the zone

necessary to achieve 1000:1 dilution.

In one instance, an unconventional WWTP that used membrane filtration technology

rather than conventional treatment with chlorine or UV disinfection was assessed.

The levels of NoV GII in shellfish sentinels near this WWTP were greater than 300

NoV particles/100 g of DD, even when dilution levels were greater than 1000:1, and

on two occasions when dilution levels exceeded 10,000:1. In seven (7) instances,

NoV levels at the plant were greater than 300 NoV particles/100g of DD. MSC levels

were similarly high, with all six (6) samples tested having MSC levels greater than

800 PFU/100g, and in one sample greater than 10,000 PFU/100g, even though

dilution levels were higher than 1000:1. This analysis demonstrates the need to assess

WWTPs with unique treatment systems on a case by case basis, since some may

perform better than conventional WWTPs at removing viruses and some may perform

significantly worse.

The overall results of FDA’s studies demonstrate a strong relationship between

increased levels of enteric viruses and MSC and decreased levels of dilution. This

trend was observed in all of the studies conducted by FDA at conventional WWTPs.

The FDA studies also suggested that certain factors, such as the quality of sewage

treatment or the time of year, may exert influences on the levels of viruses discharged

and hence the minimum level of dilution needed to ensure shellfish safety. However,

at this time FDA does not have reliable data to justify a recommended minimum




dilution less than 1000:1 or to establish any variable dilution thresholds

corresponding to and dependent on such factors. It is recognized that these criteria

could be determined by a State Shellfish Control Authority (SSCA) on a case by case

basis, where factors of WWTP performance, disinfection method, tidal flushing, and

seasonal impacts may vary. These and other factors that might influence virus levels

in the shellfish can be considered by SSCAs when assessing how best to manage

conditional growing areas based on WWTP performance. Using dilution levels lower

than 1000:1 or other alternative approaches for managing the viral risk posed by

WWTP effluents are cited in Alternate Options section (see below). However, when

there is insufficient information available for a growing area to support the use of a

lower level of dilution, the 1000:1 dilution should be employed.

Alternate Options

It is expected that the principles of this guidance shall be followed to ensure

compliance with the dilution requirements of the Model Ordinance. An alternative

minimum waste water dilution threshold value may be appropriate for situations in

which highly effective WWTP facilities reduce the viral load of the effluent, or

seasonal or geographical factors reduce the risk of viral contamination at the shellfish

growing area. Alternative options for calculating the size of the prohibited zone to mitigate the virological effects of WWTP discharges at the shellfish growing area

may be used provided that they are based on sound scientific principles that can be

verified. For example, it is reasonable to expect a potentially higher reduction in viral

load from a properly maintained wastewater treatment system employing ultraviolet

(UV) disinfection with tertiary treatment operating under optimum design flow

conditions. Regardless of the technology employed any proposed alternative

minimum threshold would need validation. MSC could potentially be used on a case-

by-case basis as the validation process (for example to validate treatment efficiency)

if demonstrated it is a successful/feasible strategy for the given location/situation

It should be noted that any alternate approach would need to consider the time

of waste transport to the shellfish harvest site. As described in this guidance in

geographic regions with large tidal amplitudes and/or swift tidal currents, the

time of waste transport to the shellfish harvest site may be the determining factor

in sizing the prohibited zone. However, there may be various strategies that

could be employed to address the time of waste transport to the shellfish

harvest site. For example, it may be reasonable to expect that if a facility

utilized a sufficiently sized containment structure (such as the equivalent to

24-hour holding for the design capacity of the plant) in the event of a

malfunction, this would allow the SSCA additional time to react to the event

and take any necessary precautions. Regardless of technology or best

management practices employed any proposed alternative strategy would need

to be validated (ie verifying that a containment structure is properly sized and

working effectively).

There are likely other alternatives in addressing the potential impact of

wastewater on shellfish growing areas and approaches in validating these




options. However, the flexibility remains with the SSCA’s to determine the

appropriate alternate option and validation process that can be verified.

References

Batz, M. B., Hoffman, S., Morris, G.J. Ranking the Disease Burden of 14 Pathogens

in Food Sources in the United States Using Attribution Data from Outbreak

Investigations and Expert Elicitation. Journal of Food Protection, Vol 75 (7):1278-

1291

Baggi, F., A. Demarta, and R. Peduzzi. (2001) Persistence of viral pathogens and

bacteriophages during sewage treatment: lack of correlation with indicator bacteria.

Res. Microbiol. 152, 743–751

Bedford, A.J., G. Williams, and A.R. Bellamy. 1978. Virus accumulation by the

rock oyster Crassostrea glomerata. Appl. Environ Microbiol. 35(6):1012-8.

Brooks, N.H. 1960. Diffusion of sewage effluent in an ocean current. In: E.A.

Pearson, editor. Proceedings of the First Conference on Waste Disposal in the Marine

Environment. New York, NY: Pergamon Press. pp. 246-267.

Burkhardt, W. III, J.W. Woods, and K.R. Calci. 2005. Evaluation of Wastewater

Treatment Plant Efficiency to Reduce Bacterial and Viral Loading Using Real-time

RT-PCR. Poster Presentation, ASM, Atlanta, GA, Annual Educational Conference.

Burkhardt, W. III, W.D. Watkins, and S.R. Rippey. 1992. Seasonal effects on

accumulation of microbial indicator organisms by Mercenaria mercenaria. Appl.

Environ. Microbiol. 58:826-31.

Burkhardt, W. III, and K.R. Calci. 2000. Selective accumulation may account for

shellfish-associated viral illness. Appl. Environ. Microbiol., Vol. 66(4): 1375-1378.

Burkhardt, W III. Calci, K. R., Watkins, W. D., Rippey S. R. and Chirtel, S. J. 2000.

Inactivation of Indicator Organisms in Estaurine Waters. Wat. Res. 34(9): 2207-

2214.

DePaola, A., J.L. Jones, J. Woods, W. Burkhardt III, K.R. Calci, J.A. Krantz, J.C.

Bowers, K. Kasturi, R.H. Byars, E. Jacobs, D. Williams-Hill, & K. Nabe. 2010.

Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey.

Appl. Environ. Microbiol. 76(9):2754-2768.

Dore, W.J. and D.N. Lees. 1995. Behavior of Escherichia coli and male-specific

bacteriophage in environmentally contaminated bivalve molluscs before and after

depuration. Appl. Environ. Microbiol. 61:2830-2834.

Dore, W.J., K. Henshilwood, and D.N. Lees. 2000. Evaluation of F-Specific RNA

bacteriophage as a candidate human enteric virus indicator for bivalve molluscan

shellfish. Appl. Environ. Microbiol. 66(4):1280-1285.

Enriquez, R., G.G. Frosner, V. Hochstein-Mintzel, S. Riedermann, and G. Reinhardt.

1992. Accumulation and persistence of hepatitis A virus in mussels. J. Med Virol.




37(3):174-9.

Goblick, G.N., Anbarchian J M,. Woods J.,, Burkhardt W. and Calci

K. 2011. Evaluating the Dilution of Wastewater Treatment Plant Effluent and Viral

Impacts on Shellfish Growing Areas in Mobile Bay, Alabama. Journal of Shellfish

Research, Vol. 30 (3), 1-9.

Havelaar, AH, M. van Olphen, and Y.C. Drost. 1993. F-specific RNA

bacteriophages are adequate model organisms for enteric viruses in fresh water.

Appl. Environ. Microbiol. 59(9):2956-2962.

Iwamoto, M., Ayers, T., Mahon, B and Swerdlow, D.L 2010. Epidemiology of

Seafood-Associated Infections in the USA. Clinical Microbiology Reveiws.

April,2010 . p399-411.

Jaykus, L., M.T. Hemard, and M.D. Sobsey. 1994. Human enteric pathogenic

viruses. In: C.R. Hackney and M.D. Pierson, editors. Environmental Indicators and

Shellfish Safety. New York, NY: Chapman and Hall. pp. 289-330.

Kapikian, AZ and Chanock RM. 1990. Norwalk Group of Virus in Virology. New

York, NY: Raven Press Ltd. pp. 671-693.

Kohn, et al. 1995. An Outbreak of Norwalk Virus Gastroenteritis Associated with

Eating Raw Oysters, Implications of Maintaining Safe Oyster Beds. JAMA.

Lumsden, L.L. Hassetline, H. E., Leake, J.P. and Veldee, M. V. A Typhoid Fever

Epidemic Caused by Oyster-Borne Infection (1924-5). Supplement No 50 to the

Public Health Reports. Washington Government Printing Office 1925.

Liu, OC, Seraichekas, HR, Murphy, BL. 1966. Viral Pollution of Shellfish, I: Some

Basic Facts of Uptake. Proc. Soc. Exp. Biol. Med. 123:481-487.

Love, D.C., Lovelace, G.L., & Sobsey, M.D. 2010. Removal of Escherichia coli,

Enterococcus fecalis, coliphage MS2, poliovirus, and hepatitis A virus from oysters

(Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) by depuration.

Int.J.Food Microbiol., 143, (3) 211-217

Maalouf, F. Schaeffer, J., Parnaudeau, S., Le Pendu, J.. Atmar, R., Crawford, S.E. &

Le Guyader, F.S. (2011) Strain-dependent Norovirus bioaccumulation in oysters.

Applied and Environmental Microbiology 77(10): 3189

Metcalf, T.G., B. Mullin, D. Eckerson, E. Moulton, and E.P. Larkin.

1979. Accumulation and depuration of enteroviruses by the soft-shelled clam, Mya

arenaria. Appl. Environ. Microbiol. 38(2):275-82.

Nash, J.D. 1995. Buoyant Discharges into Reversing Ambient Currents, MS Thesis,

DeFrees Hydraulics Laboratory, Cornell University, Ithaca, NY.

Scallan, E., Hoekstra, R.M. Tauxe, R. V et al. Foodborne Illness Acquired in the

United States – Major Pathogens. Emerging Infectious Diseases Vol17. No1, January

2011. www.cdc.gov/eld


http://www.cdc.gov/eld



Seraichekas, H. R., D. A. Brashear, J. A. Barnick, P. F. Carey & O. C. Liu. 1968.

Viral deputation by assaying individual shellfish. Appl. Microbiol. 16:1865-1871.

Shieh, C. Y., R.S. Baric, J.W. Woods, and K.R. Calci. 2003. Molecular surveillance

of enterovirus and Norwalk-like virus in oysters relocated to a municipal-sewage-

impacted Gulf estuary. Appl. Environ. Microbiol. 69(12):7130-7136.

Sobsey, M.D., A.L. Davis, and V.A. Rullman. 1987. Persistence of hepatitis A virus

and other viruses in depurated eastern oysters. In: NOAA, editor. Proceedings,

Oceans ’87. Halifax, Nova Scotia: NOAA. 5:1740-1745.

Public Health

Significance:

The public health purpose of this guidance is to provide the scientific basis and

recommendations for determining appropriately sized Prohibited Areas (closure

zones) around waste water treatment plants (WWTP). Section II, Chapter IV @ .03

(5) currently mandates that a prohibited zone be established, but there is no specific

guidance information on how to calculate the size of the prohibited zone to ensure that

microbiological pathogens (particularly viruses) from WWTP do not adversely impact

the growing area at the time of harvest. It is expected that this guidance will provide

all ISSC stakeholders with better information on which to make informed,

scientifically based decisions

Cost Information

(if available):


Proposal No. 13-119-L

2013 Task Force I – Proposal No. 13-119-L - Page 1 of 3




Growing Area


Administrative

Submitter: Laboratory Methods Review & Quality Assurance Committee Patti Fowler, Chairperson

Affiliation: Interstate Shellfish Sanitation Conference

Address

209 Dawson Road Suite 2

Columbia, SC 29223-1740

Phone: 803-788-7559

Fax: 803-788-7576

Email: [email protected]

Proposal Subject: Revisions to Chapter III. Requirements for the Authority

Specific NSSP

Guide Reference:

2011 NSSP Guide Section II. Model Ordinance Chapter III. Laboratory

Text of Proposal/

Requested Action

@.02 Methods.

A. Microbiological. Methods for the analyses of shellfish and shellfish growing or harvest waters shall be:

(1) The Approved NSSP Methods validated for use in the National Shellfish Sanitation Program under Procedure XVI. of the Constitution,

Bylaws and Procedures of the ISSC and / or cited in the Guidance

Documents Chapter II. Growing Areas .11 Approved National Shellfish Sanitation Program Laboratory Tests.

(2) When there is an immediate or ongoing critical need for a method and no Approved NSSP Method exists, the following may be used:

(a) A validated AOAC, BAM, or EPA method; (b) An Emergency Use Method pursuant to .02 D. (1) and (2) below.

B. Chemical and Physical. Methods for the analyses of shellfish and shellfish harvest waters shall be:

(1) The Approved NSSP Methods validated for use in the National Shellfish Sanitation Program under Procedure XVI. Of the Constitution, Bylaws and Procedures of the ISSC and cited in the Guidance Documents

Chapter II. Growing Areas .11 Approved National Shellfish Sanitation

Program Laboratory Tests. Methods for the analysis of shellfish and

shellfish growing or harvest waters shall: (a) Be the current AOAC or APHA method for all physical and chemical measurements; and

(b) Express results of all chemical and physical measurements in standard units, and not instrument readings.

(2) Results shall be expressed for chemical and physical measurements in standard units and not instrument readings. (2)(3) When there is an immediate or ongoing critical need for a Method and no Approved NSSP Method exists, the following may be used:



mailto:[email protected]



C. Biotoxin. Methods for the analyses of shellfish and shellfish harvest waters shall be:

(1) The Approved NSSP Methods validated for use in the national Shellfish Sanitation Program under Procedure XVI. Of the Constitution,

Bylaws and Procedures of the ISSC and cited in the Guidance Documents

Chpater II. Growing Areas .11 Approved National Shellfish Sanitation Program Laboratory Tests. The current AOAC and APHA methods used

in the bioassay for paralytic shellfish poisoning toxins; and

(2) The current APHA method used in the bioassay for Karenia brevis toxins; or

(3) Approved NSSP Methods validated for use under Procedure XVI. of the Constitution, Bylaws and Procedures of the ISSC and/or cited in the Guidance Documents Chapter II. Growing Areas .11 Approved National

Shellfish Sanitation Program Laboratory Tests.

(4)(2) When there is an immediate or ongoing critical need for a method and no Approved NSSP Method exists, the following may be used:


D. Emergency Use Methods. (1) When there is an immediate or critical need and no Approved NSSP Method exists, an unapproved or non-validated method may be used for a specific purpose provided that:

(a) The appropriate FDA Regional Office is notified within a reasonable period of time regarding the method employed; and

(b) The ISSC Executive Board is notified within a reasonable period of time regarding the method employed.

(2) When it is necessary to continue the use of the emergency method employed under D. (1) beyond the initial critical need, then the following minimum criteria shall be provided to the ISSC Executive Board for

interim approval:

(a) Name of Method. (b) Date of Submission. (c) Specific purpose or intent of the method for use in the NSSP. (d) Step by step procedure including equipment, reagents and safety requirements necessary to run the method. (e) Data generated in the development and/or trials of the method and/or comparing to approved methods if applicable.

(f) Any peer reviewed articles detailing the method. (g) Name of developer(s) or Shellfish Control Authority submitter. (h) Developer/submitter contact information.

(3) Within two (2) years of Executive Board interim approval of the Emergency Use Method, the entire Single Lab Validation Protocol should be submitted. The Laboratory Methods Review Committee will report to

the Executive Board on the status of the Single Lab Validation Protocol

data submission.




Public Health

Significance:

This revision to Chapter III. Laboratory is necessary to clarify and guide users to the

location within the Guidance Documents that lists the approved NSSP laboratory tests

in .11 Approved NSSP Laboratory Tests. All approved laboratory tests are now listed in Table .11 Approved NSSP Laboratory Tests with the Guidance Document.

Cost Information

(if available):






Growing Area


Administrative

Submitter: Thomas Howell

Affiliation: Spinney Creek Shellfish, Inc.

Address: 27 Howell Drive

Eliot, ME, 03903

Phone: (207) 439-2719

Fax: (207) 439-7643

Email: [email protected]

Proposal Subject: Male-specific Coliphage Method for Quahogs (M. mercenaria)

Specific NSSP

Guide Reference:

NSSP Guide Section IV Guidance Documents Chapter II Growing Areas

.11 Approved Limited Use Methods for Microbiological Testing

Text of Proposal/

Requested Action

This submission presents the ‘Male-specific Coliphage method for Quahogs (M.

mercenaria)’ for consideration as an approved limited use method for

microbiological testing. At the 2009 ISSC, the ‘Modified Double Agar Overlay

Method for Determining Male-specific Coliphage in Soft-shelled Clams and

American Oysters’ was accepted as an approved limited use method for

microbiological testing for re-opening growing areas after emergency closures due

to sewage spills. SLV work with quahogs has demonstrated comparable

performance characteristics as with soft-shelled clams and American oysters.

The requested action is to include quahogs in the footnote for MSC along with soft-

shelled clams and American oysters in NSSP Guide Section IV Guidance

Documents Chapter II Growing Areas .11 Approved Limited Use Methods for

Microbiological Testing.

Public Health

Significance:

The MSC method for quahogs was used recently by the State of New Jersey to re-

open growing areas after the devastating effects of Superstorm Sandy. Increasingly,

enumeration of male-specific coliphage (MSC) in soft-shelled clams, American

oysters, and quahogs is needed in the NSSP to assess viral contamination in

molluscan shellfish harvested from growing areas where fecal coliform levels in

both water quality and shellfish meats may be misleading. MSC is a specialized

indicator of viral sewage contamination, which is substantially more meaningful

than fecal coliform or E. coli in evaluating the safety of shellstock harvested from

growing areas potentially impacted by treated and partially treated wastewater.

Cost Information

(if available):

This method for the enumeration of male-specific coliphage in soft-shelled clams,

American oysters, and quahogs is inexpensive, easy to perform, and rapid, providing

results within 24 hours. The cost of laboratory glassware, plastic-ware, agars, and

reagents is approximately $25 per shellfish sample. In a well-equipped laboratory,

the method requires 6 hours of time from initiating host to pouring plates. Hands on

technician time to perform this test is significantly less on the order of 1-4 hours per

test depending upon how many tests are done per day. The most expensive piece of

equipment is a refrigerated centrifuge plus rotor, which costs approximately

$12,000. There are no special skill sets required beyond those required to operate a

state-approved shellfish laboratory under the NSSP.


mailto:[email protected]

1

Method Application and Single Lab Validation Checklist For Acceptance of a Method for Use in the NSSP

The purpose of single laboratory validation in the National Shellfish Sanitation Program (NSSP) is to ensure that the analytical method under consideration for adoption by the NSSP is fit for its intended use in the Program. A Checklist has been developed which explores and articulates the need for the method in the NSSP; provides an itemized list of method documentation requirements; and sets forth the performance characteristics to be tested as part of the overall process of single laboratory validation. For ease in application, the performance characteristics listed under validation criteria on the Checklist have been defined and accompany the Checklist as part of the process of single laboratory validation. Further a generic protocol has been developed that provides the basic framework for integrating the requirements for the single laboratory validation of all analytical methods intended for adoption by the NSSP. Methods submitted to the ISSC LMR Committee for acceptance will require at a minimum 6 months for review from the date of submission.

Name of the New Method Male-specific Coliphage for Quahogs (M. Mercenaria) Name of the Method Developer

Thomas Howell, Spinney Creek Shellfish, Inc.

Developer Contact Information Spinney Creek Shellfish, Inc. 27 Howel Drive Eliot, ME 03903 (207) 439-2719 [email protected]

Checklist Y/N Submitter Comments A. Need for the New Method Clearly define the need for which the method has been developed.

Y

What is the intended purpose of the method?

Y

Is there an acknowledged need for this method in the NSSP?

Y

What type of method? i.e. chemical, molecular, culture, etc.

Y Culture method for Male-specific Coliphage in Quahogs (M. Mercenaria)

B. Method Documentation 1. Method documentation includes thefollowing information:

Method Title Y Method Scope Y References Y Principle Y Any proprietary aspects N Equipment required Y Reagents required Y Sample collection, preservation and storage requirements

Y


2

Safety requirements Y Clear and easy to follow step-by-step procedure

Y

Quality control steps specific for this method

Y

C. Validation Criteria 1. Accuracy / Trueness Y 2. Measurement uncertainty Y 3. Precision characteristics

(repeatability) Y

4. Recovery Y 5. Specificity NA 6. Working and Linear ranges Y Working Range 7. Limit of detection Y 8. Limit of quantitation / Sensitivity Y 9. Ruggedness Y

10. Matrix effects NA Matrix effects were observed and modifications made to the MSC method during SLV work with soft-shelled clams and American oysters in 2008-2009. These same modifications are employed in this mehtod for quahogs. No matrix effects are anticipated

11. Comparability (if intended as asubstitute for an established method accepted by the NSSP)

NA

D. Other Information 1. Cost of the method Y 2. Special technical skills required to

perform the method Y

3. Special equipment required and associated cost

Y

4. Abbreviations and acronyms defined

Y

5. Details of turn around times (time involved to complete the method)

Y

6. Provide brief overview of the qualitysystems used in the lab

Y

Submitters Signature Date: Submission of validation data and draft method to committee

Date:

Reviewing members:

Accepted Date:


3

Recommendations for further work Date:

Comments:


4

Single Laboratory Validation (SLV) Protocol

For Submission to the Interstate Shellfish Sanitation Conference (ISSC)

For Method Approval

Section A. Justification for New Method

Name of the New Method - Male-specific Coliphage (MSC) for Quahogs.

Specify the Type of Method - Culture Method/Double Agar Overlay Method

Name of Method Developer - Thomas Howell, Spinney Creek Shellfish, Inc.

Developer Contact Information – Spinney Creek Shellfish, Inc. 27 Howell Drive Eliot, Maine 03903 (207) 439-2719 (207) 439-7643 FAX [email protected]

Date of Submission – November 8, 2013

Purpose and Intended Use of the Method.

The primary purpose and intended use of this method in the NSSP is for re-opening growing areas after emergency closures due to sewage spills. This method has been used recently to re-open growing areas after the devastating effects of Superstorm Sandy by the State of New Jersey. The method presented in this document is the same as that modified and validated for soft-shelled clams and American oyster at the 2009 ISSC in Manchester, NH. Additionally, this method can be used to verify and optimize viral depuration/relay strategies used to reduce viral contamination in quahogs harvested from growing areas impacted by wastewater treatment plant (WTP) outfall.

Need for the New Method in the NSSP, Noting Any Relationships to Existing Methods.

Fecal coliforms (FC), a bacterial indicator, are used for process validation for conventional depuration processes. In growing areas impacted by moderate or low-level non-point source contamination, conventional depuration methods using FC for process validation are adequate, well proven, and widely accepted by the scientific and public health community. Statistical analysis of FC samples, collected during water quality monitoring, are used to determine growing area classification. Limits on the geometric mean and 90th percentile are considered adequate to protect public health from the risks of viral contamination in areas not impacted by sewage and WTP pollution. However, in growing areas impacted by treated sewage, the relationship between bacterial and viral contamination can be substantially altered by the differential inactivation rates of chlorination and other disinfection methods on bacteria and


5

viruses. This MSC method is needed in the NSSP to evaluate viral contamination in molluscan shellfish harvested from growing areas where FC levels in both water quality and shellfish meats may be misleading. MSC is a specialized indicator of viral contamination, which is substantially more meaningful than FC in evaluating the safety of shellstock harvested from growing areas potentially impacted by treated and partially treated wastewater. Much work has been done to demonstrate that the MSC method is particularly useful and highly advantageous over FC for evaluating the efficacy of viral depuration and viral relay processes in soft-shelled clams. Continuing work is being conducted to assess the usefullness of this method for evaluating the efficacy of viral depuration and viral relay processes for American oysters and quahogs.

Method Limitations and Potential Indications of Cases Where the Method May Not Be Applicable to Specific Matrix Types.

The MSC method described here has been previously validated for soft-shelled clams and American Oysters and is currently being evaluated for quahogs. Further SLV work is needed to evaluate different matrix types / other species of molluscan shellfish.

Other Comments.

SLV work strongly suggests that this modified MSC method is appropriate (fit for purpose) for applications in Quahogs in addition to Soft-shelled clams and American oysters where a regulatory limit of 50 PFU/100gram has been established.


6

Section B. Method Documentation

Modified Double Agar Overlay Method for Determining Male-specific Coliphage

In Soft-shelled Clams, American Oysters, and Quahogs (M. mercenaria) Nov 2013

This method for determining levels of male-specific coliphage in quahog meat is based on the method described by DeBartolomeis and Cabelli1,2. FDA had refined the method for oyster and hard clam meats as described in the workshop instructions, Male-specific Bacteriophage (MSB) Workshop, conducted in Gloucester, Massachusetts on March 9-12, 20043. This original FDA (2004) method was submitted as ISSC Proposal 05-114. This method was modified again in 2008-2009 by Spinney Creek Shellfish to improve viral recovery and sensitivity for soft-shelled clams and American oysters. Modification of the FDA (2004) Method Spinney Creek Shellfish, Inc. (SCS) further refined these procedures for soft-shelled clam and oyster meat in 2006. In this work and in parallel work conducted by Mercuria Cumbo of the Maine Department of Marine Resources, it was observed that the extraction protocol was inadequate. The supernatant produced when soft-shelled clams and some oysters were processed was opaque and creamy while the pellet was loose and indistinct. Subsequent re-washing of the pellets in growth broth, re-processing, and re-plating showed significant levels of MSC left in the pellet, indicating poor recovery. The problem was solved by; eluting the shellfish meats with growth broth (2:1), and increasing the blending time to 180 seconds. This modification, based on EU methodology (ISO 10705-4), resulted in a clear supernatant and a distinct, firm pellet. Further experimentation and subsequent validation work confirmed that this elution approach works very well. SLV validation work conducted by (SCS) in 2009 resulted in further modification of the method to increase the limit of quantitation/sensitivity (LOQ). This increase in LOQ was achieved by plating an increased amount of supernatant (25ml) and using 10 plates. This same modified method is used for quahogs in the SLV application. A. Apparatus and Materials. Equipment and Materials for Collection and Transport of Shellfish Samples: 4 mil plastic bags Labels Cooler Gel Packs Temperature Control Blank Laboratory Equipment: Centrifuge with rotor for 50 ml conical (or larger) tubes, 9000 x g performance capability, 4°C Water bath, 50-52°C Air Incubator, 35-37°C Balance Stir plate and magnetic stirring bars, sterile


7

Mini vortexer Blender Autoclave, 121°C Refrigerator, 0–4° C Freezer, -20°C Thermometers, range -20–121°C pH meter Erlenmeyer flasks, 1 L and 2 L Graduated cylinders, 100 ml, 500 ml and 1000 ml 600ml and 3000ml beaker 500 ml jars, autoclavable with caps Inoculating loops (3 mm in diameter or 10 FL volume) Bacti-cinerator Sterile swabs Sterile, disposable filters, 0.22 or 0.45 µm pore size Syringes, sterile disposable; 5, 10 or 20 ml Scrub brushes, sterile Knives, sterile Blender jars, sterile Sterile plastic cups 250 ml Pipets- 2ml, 5 ml, 10 ml Pipet-aid Micro-Pipettors, 100 µL, 200 µL, 1000 µL, 2500 µL Micro-Pipet tips 200 µL, 1000 µL, 2500 µL Pipetor Stand Centrifuge tubes, sterile disposable 50 ml or larger Petri dishes, sterile disposable 100 x 15 mm Petri dish racks Test tubes 16 x 100 mm (for soft agar) Test tubes 16 x 150 mm, with screw caps Test tube racks--size to accommodate tubes Freezer vials, sterile 30 ml with screw caps Baskets with tops to hold freezer vials Parafilm tape Aluminum foil Reagents: Reagent water Glycerol- sterile Ethanol, 70% or laboratory disinfectant Calcium chloride, 1M Mineral oil Antibiotic stocks: Ampicillin sodium salt (Sigma A9518) Streptomycin sulfate (Sigma S6501) Streptomycin and Ampicillin stock solutions (50 µg/ml each). Note: Antibiotics must always be added to liquids and media after these have been autoclaved and cooled.


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Media: Bottom Agar DS Soft Agar Growth Broth Bacterial Host Strain: E.coli Famp . E. coli HS(pFamp)RR (selected by Dr. Victor J. Cabelli, University of Rhode Island, Kingston, RI, USA, frozen stock ATCC # 700891). MSC (Coliphage) Stock: Type Strain - MS2, ATCC # 15597 B. Media Composition. Bottom Agar: Tryptone 10.0 g Dextrose 1.0 g NaCl 5.0 g Agar 15.0 g DI water 990 ml Final pH 6.7 ± 0.2 at 25°C 1. With gentle mixing, add all the components to 990 ml of dH2O in a 2000 ml flask. Dissolve,

heat until clear and boiling started. 2. Sterilize at 121°C ± 2°C for 15 minutes. 3. Temper to 50°C in the water bath. 4. Add 5 ml of Streptomycin sulfate/Ampicillin solution, aseptically to the flask (50 µg/ml each

in final) and mix. Transfer to 2 – 500ml sterile jars (easier to pour plates from jars). 5. Pipet (or pour) 15 ml aliquots aseptically into sterile 100 x 15 mm Petri dishes and allow the

agar to harden. Tip Petri dish lids off slightly to reduce condensation. 6. Store bottom agar plates inverted at 4°C and warm to room temperature for 1 hour before

use. 7. Plates stored sealed at 4°C can be used up to 6 weeks. Streptomycin sulfate/Ampicillin Solution: 1. Dissolve 0.5g of streptomycin sulfate and 0.5g of ampicillin in 50 ml of dH2O with a sterile

100 ml graduated cylinder in sterile 600 ml beaker with sterile stir bar. 2. Stir for 2 to 3 minutes, no heat. 3. Filter through sterile 0.22 µm filter. 4. Store in 5 ml aliquots in sterile 30 ml capped freezer vials at -20°C for up to one year. Label

and date. 5. Allow to come to room temperature before adding and mixing in tempered bottom agar at

50°C.


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DS Soft Agar: Tryptone 10.0 g Dextrose 1.0 g NaCl 5.0 g 1M CaCl2 0.5 ml Agar 7.0 g DI water 500 ml Final pH 6.7 ± 0.2 1. With gentle mixing, add all the components to 500 ml of dH2O in a 1000 ml flask. 2. Bring flask contents to a boil. 3. Dispense in 2.5 ml aliquots into 16 x 100 ml tubes, cover and freeze (-20°C) for up to three

months. 4. Sterilize prior to use at 121°C ± 2°C for 15 minutes, then temper to 50-52°C for no longer

than 2 hours 1M CaCl2 Solution: 1. Add 11.1 g of CaCl2 anhydrous (FW 111.0, Dihydrate FW 147) to 100 ml 2. dH2O in a screw top bottle and dissolve or use prepared from VWR. 3. Sterilize by autoclaving at 121°C for 15 minutes. 4. Store up to three months at 4°C. 5. Use at room temperature. Growth Broth: Tryptone 10.0 g Dextrose 1.0 g NaCl 5.0 g DI water 1000 ml 1. With gentle mixing, add all the components to 1000 ml of dH2O water in a 2000 ml flask. 2. Dissolve and dispense into sterile screw top containers. 3. Sterilize at 121°C ± 2°C for 15 minutes. 4. Store for up to three months at 4°C. Storage Slants: Tryptic Soy Agar. C. Storage and Propagation of Host Strain, E. coli Famp. Storage: 1. Lab stock culture – Frozen at – 80°C indefinitely (most desirable method) in broth culture

containing 10% glycerol under no selective pressure. Selective pressure is reapplied when the culture is retrieved, by streaking onto Bottom Agar plates containing the two antibiotics.

2. Long-term working stock culture – Grown tryptic soy agar slant with sterile mineral oil overlay under no selective pressure and stored at room temperature in the dark for up to 2 years.

3. Long-term working stock – 6-hour grown tryptic soy agar slant and deep stab with sterile mineral oil overlay containing the two antibiotics, Ampicillin and Streptomycin (least desirable method).


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4. Short-term working stock culture - Grown Bottom Agar streak plate stored at 4°C up to 3 weeks.

5. Short-term working stock culture – Grown in Growth broth and used within 6-12 hours (same day).

Glycerol Solution, 10%: Add 9 ml of distilled water to 1 ml of undiluted glycerol. Autoclave resulting 10% glycerol solution at 121°C for 15 minutes and use at room temperature. For storage, add 1/5th volume of 10% glycerol solution, let stand for 30 minutes, dispense 1 ml aliquots in 2 ml cryo-vials and store at -70 to –80°C (best) or at –20°C. Propagation: 1. Vortex to aerate 10 ml of Growth Broth medium tempered to 35 – 37°C just prior to

inoculation. 2. Transfer host strain to Growth Broth using sterile swab to collect material from several

colonies off grown Bottom Agar streak plate and warmed to room temperature. 3. Gently shake to mix, then incubate at 35–37°C for 4-6 hours (turbidity=107cells/ml; O.D at

540nm=0.4). 4. Once turbidity is observed, use of the host strain broth culture (log-phased growth) may

commence (following initial inoculation and mixing, do not shake or mix the host strain broth culture). D. Control Plates. 1. Negative Control - Add 2.5 ml of Growth Broth and 0.2 ml host to the 2.5 ml DS Soft Agar

tube. 2. Positive Control - Make serial dilutions using growth broth of the concentrated MS2 control

(to grow approximately 50-100 PFU per 2.5 ml), and add 2.5 ml of appropriate MS2 dilution and 0.2 ml of host to 2.5 ml DS Soft agar.

E. MSC Density Determinations in Soft Shelled Clam, American Oyster, and Quahog Tissues. Sample Requirements. Samples of shellstock and shucked meats are held under dry refrigerated conditions at 1–4°C. Samples must be comprised of a representative number of animals (12 to 15). Samples are analyzed within 24 hours of collection. Animals with broken shells or animals that appear dead are discarded. Sample collection bags must be properly identified with lot #, date and time of collection, collection location and collector’s initials. Preparation of Shellfish for Analysis. Using soap and water, analyst’s hands are thoroughly scrubbed and rinsed. Using a sterile brush, shells of whole animals are scrubbed under running potable water to remove loose material from the shells. Shellfish then are placed on a clean paper towel or in an open weave basket to dry. Scrubbed, drying animals should not come in contact with each other. Once the shells of washed shellfish are dry, analysts wash their hands thoroughly with soap and water, then rinse their hands with 70% alcohol and allow to air dry. Shellfish are shucked and the meats and liquors are saved into a sterile 250 ml cups.


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Direct Analytical Technique for Soft Shelled Clams, American Oysters, and Quahogs. For each soft shelled clam, American oyster, or quahog sample, ten (10) Bottom Agar plates and ten (10) 2.5 ml DS Soft Agar tubes are prepared. Use a 4 to 6 h culture of host strain, E. coli Famp. Always begin analyses with a negative control (blank) plate and finish analyses with a positive control plate followed by a second negative control plate. 1. Shuck 12 soft shelled clams, American oysters, or quahogs into sterile 250 ml cup, tare and

add to sterile blender. To make a 1:2 (wgt:vol) elution with growth broth eluent using twice the volume of the shellfish. Add to blender with sample. Homogenize by blending for 180 seconds at high speed.

2. Immediately weigh 33.0 g of homogenate from each sample into labeled sterile 50 ml centrifuge tubes after blender has stopped before foam separation can occur.

3. Centrifuge each sample for 15 min. @ 9,000-10,000 x g; 4°C. 4. Pipette off and weigh the supernatant in a new sterile 50 ml centrifuge tube. 5. Allow the supernatant to warm to RT (approximately 20-30 minutes). 6. Shake or vortex the supernatant. 7. Gently pipette 200 µL of log phase host strain, E. coli HS(pFamp)RR using 200 µL micro

pipettor and a 200 µL pipet tip, then pipette 2500 µL aliquot of supernatant using the 2500 µL micro pipettor and a 2500 µL pipet tips, to 2.5 ml DS Soft agar tube (tempered to 52°C).

8. Once E. coli Famp is added to the mixture do not shake, only gently mix contents by rolling the tube between palms.

9. Overlay the 5.2 ml onto a Bottom Agar plate containing Streptomycin and Ampicillin (50 g/ml final concentrations). Drag the mixture into a clear area and gently swirl the plates to spread sample and agar mixture.

10. Allow plates to set then inverted and incubated for 16 - 20 hours at 35- 37°C. Calculations of Results Total number of MSC (N) x Weight of supernatant extracted (Ws) x 100 = Total supernatant plated (25gm) grams of sample used (11gm) N x Ws x 100 = (0.364)(N)(Ws) = PFU of MSC/100 gm 25 gm 11 gm Example: Clam/Oyster plate counts - 13, 23, 12, 16, 12, 18, 17, 21, 19, 17 and 27.5 g supernatant. Result = (0.364)*(168MSC)(27.5gm) = 1681 PFU of MSC/100 gm *0.364=100/(25 x 11) F. Sample Collection and Storage. 1. Record all pertinent information on the collection form. 2. During transportation store samples in a cooler at 0 to 10°C. 3. At laboratory, store samples in a refrigerator at 0 to 4 °C. 4. Maximum holding times for shellfish samples is up to 24 hours.


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G. Quality Assurance. 1. Positive and negative control plates are run with MSC analyses each day. 2. Media sterility checks are made per batch and records are maintained. 3. Media log book is maintained (pH, volume, weights of each components, lot numbers, etc.). 4. An intra- and inter-laboratory performance program is developed. 5. Circular zones of clearing (typically 1 to 10 mm in diameter) in lawn of host bacteria after

16- 20 hours of incubation are counted as plaques. (Count the number of plaques on each plate.)

6. MSC determinations are reported as plaque forming unit (PFU) per 100 grams. 7. The desired range for counting is 0 to 100 PFU per plate. If the count exceeds the upper

range or if the plaques are not discrete, results should be recorded as “too numerous to count” (TNTC) or >10,000 PFU of MSC/100gm.

8. Temperatures incubators are checked twice daily (at least 4 hours apart) to ensure operation within the stated limits of the method, and results are recorded in a logbook.

9. Check thermometers at least annually against a NIST-certified thermometer. 10. Calibrate the balance monthly using ASTM-certified Class 1 or 2 or NIST Class S reference

weights. 11. Laboratory analysts adhere to all applicable quality control requirements set forth in the most

recent version of FDA's Shellfish Laboratory Evaluation Checklist. 12. Calibration of micro-pipettors needs to be checked quarterly and records kept. Micro-

pipettors used for handling MSC control and transferring host cells need to have a barrier tip or be dedicated to the specific use to prevent contamination

H. Safety. Samples, reference materials, and equipment known or suspected to have Coliphage attached or contained must be sterilized prior to disposal.

I. Technical Terms. °C - degrees Celsius µL - microliter g - gram L - liter M - molar ml - milliliter rpm - revolutions per minute Ave. - average MSC - Male-specific Coliphage, Male-specific Bacteriophage, F+ Bacteriophage NIST - National Institute of Standards and Technology PFU - plaque forming units RT - room temperature TNTC - too numerous to count LOD - Limit of Detection LOQ - Limit of Quantitation Host Strain - E.coli Famp bacteria (E.coli HS(pFamp)RR) Male-specific Coliphage - Viruses that infect coliform bacteria only via the F-pili. Plaque - Clear circular zones (typically 1 to 10 mm in diameter) in lawn of host cells after

incubation.


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References: 1. Cabelli, V.J. 1988. Microbial indicator levels in shellfish, water, and sediments from

the upper Narragansett Bay conditional shellfish-growing area. Report to the Narragansett Bay Project, Providence, RI.

2. DeBartolomeis, J. and V.J. Cabelli. 1991. Evaluation of an Escherichia coli host strain for enumeration of F male-specific Coliphages. Appl. Environ. Microbiol. 57(4):1201-1205.

3. U.S. Food and Drug Administration. 2004. Male-specific Coliphage (MSC) Workshop, conducted in Gloucester, Massachusetts on March 9-12, 2004.

Other Information: This method for the enumeration of male-specific coliphage in soft-shelled clams, American oysters, and quahogs is inexpensive, easy to perform, and rapid, providing results within 24 hours. The cost of laboratory glassware, plastic-ware, agars, and reagents is approximately $25 per shellfish sample. In a well equipped laboratory, the method requires 6 hours of time from initiating host to pouring plates. Hands on technician time to perform this test is significantly less on the order of 1-4 hours per test depending upon how many tests are done per day. The most expensive piece of equipment is a refrigerated centrifuge plus rotor, which costs approximately $12,000. There are no special skill sets required beyond those required to operate a state-approved shellfish laboratory under the NSSP.


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C. Validation Criteria Preliminary Studies A master spike determination experiment was run before other SLV work was performed to evaluate the planned routine for the spike determinations. In previous SLV work with soft-shelled clams and oysters, viral clumping was identified as a problem when the master spike was evaluated using growth broth and then compared to determination of MSC levels in the soft-shelled clam and oyster matrix. The spike determination was lower than the spiked samples of clean shellfish suggesting a negative recovery (t